[ARTICLE WITHDRAWN] MicroRNA-539 Inhibits the Epithelial-Mesenchymal Transition of Esophageal Cancer Cells by Twist-Related Protein 1-Mediated Modulation of Melanoma-Associated Antigen A4.

This article has been withdrawn at the request of the publisher in December 2020.

INTRODUCTION

Esophageal cancer is one of the most aggressive digestive tract tumors, and esophageal squamous cell carcinoma (ESCC) is one of the most common causes of cancer-associated mortality, which comprises more than 90% of esophageal cancer1–3. In spite of the advances in surgical techniques and perioperative management in recent years, mortality from ESCC has not decreased significantly4,5. Since early ESCC is usually asymptomatic, most ESCC are diagnosed at advanced stages, along with local invasion and lymph node metastasis (LNM)6,7. Yet the mechanisms contributing to ESCC carcinogenesis are poorly understood.

MicroRNAs (miRs), with a length of 20–24 nucleotides, were widely studied in cell development, differentiation, migration, and invasion in various cancer types including esophageal cancer8–10. For instance, miR-203 is reported to repress tumor growth and invasion in esophageal cancer11, and miR-150 is involved in poor prognosis in ESCC by targeting zinc finger E-box-binding homeobox 1 (ZEB1)12. Previous studies have reported that miR-539 has a tumor suppressor role in several cancers, such as thyroid cancer13 and prostate cancer14. However, the functional role of miR-539 in esophageal cancer is far less understood.

Accumulating evidence has demonstrated that miRs play key roles in cell development and differentiation by mediating the posttranscriptional regulation of protein-coding genes15,16. Twist-related protein 1 (TWIST1), a basic helix–loop–helix transcription factor, has been reported to be correlated with multiple cancers, such as renal cell carcinoma, colorectal cancer, prostate cancer, and so on17–19. It is also known as an essential inducer of epithelial–mesenchymal transition (EMT)20. Currently, bioinformatics screening is a potentially cost-effective method to screen the possible target gene of miRs. With the help of TargetScan (http://www.targetscan.org), TWIST1 was identified as a putative target gene of miR-539. A previous study once claimed that TWIST1 could regulate melanoma-associated antigen (MAGE) A4 expression, suggesting the potential association between TWIST1 and MAGEA421. Whether TWIST1 is a target of miR-539 and the possible association between TWIST1 and MAGEA4 in esophageal cancer therefore urgently need to be fully investigated.

In our study, we aimed to identify the specific role of miR-539 in human ESCC cells. The mechanism by which miR-539 regulates EMT as well as the involvements of TWIST1 and MAGEA4 were also investigated. All of our efforts will provide a theoretical basis and new insights for the treatment of ESCC.

MATERIALS AND METHODS

Cell Culture

Human ESCC TE3 cells, obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan), were grown in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen, Carlsbad, CA, USA) in a humidified incubator with 5% CO2 at 37°C22.

miR Transfection

Synthetic miR-539 mimics and scrambled negative control RNA (miR-NC) were purchased from GenePharma (Shanghai, P.R. China). TE3 cells were seeded in six-well plates and cultured at 37°C. On the following day (approximately 70% confluence), cells were transfected with Lipofectamine 2000 reagent (Invitrogen) on the basis of the manufacturer’s information. In each well, 100 pmol of miR-539 mimics or miR-NC was used. The expression level of miR-539 was evaluated by quantitative reverse transcription (qRT)-PCR23.

Stable Expression of TWIST1 or MAGEA4

Full-length wild-type (wt) TWIST1 or MAGEA4 gene was cloned into the retroviral pBABE-puro vector (pB; Addgene, Cambridge, MA, USA) to generate pB-TWIST1 or pB-MAGEA4. Empty pBABE-puro vector was referred to as pB-NC. For viral assembly, the plasmids were transfected into 293T cells (American Type Culture Collection, Manassas, VA, USA) along with pCL packaging vector using Lipofectamine 2000 reagent. After 48 h of incubation, the viral supernatants (pB-NC, pB-TWIST1, and pB-MAGEA4) were harvested and were respectively transfected into TE3 cells using 8 μg/ml polybrene. Forty-eight hours later, the stable TWIST1- or MAGEA4-expressing cell lines as well as pB-NC-infected cell line were obtained after selection using puromycin (2 μg/ml) for 10 days24.

Lentivirus-Mediated Stable TWIST1 or MAGEA4 Knockdown

Short hairpin RNAs (shRNAs) against wt TWIST1 or MAGEA4 as well as shRNAs carrying a nontargeting sequence (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were inserted into pSicoR vector (Addgene) to generate sh-TWIST1, sh-MAGEA4, or sh-NC. Plasmids were transfected into 293T cells for the viral assembly along with the third-generation packaging mix (Applied Biological Materials, Richmond, BC, Canada) using Lipofectamine 2000 reagent. The virus was collected 48 h after transfection and concentrated via ultracentrifugation using an ultraspeed centrifuge (Beckman Coulter, Brea, CA, USA). Each virus production was concentrated into 100 μl of phosphate-buffered saline (PBS) and stored at −80°C. The virus titer was determined according to the protocol described by Sastry et al.25. The resultant viruses were respectively transfected into TE3 cells using 8 μg/ml polybrene at a multiplicity of infection (MOI) of 30.

Western Blot Analysis

The cells were washed twice with PBS and then lysed with 1× sodium dodecyl sulfate (SDS) loading buffer [50 mM Tris-Cl (pH 6.8), 100 mM dithiothreitol (DTT), 2% SDS, 10% glycerol, and 0.1% bromophenol blue]. The protein samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transfer. Immunoblotting was carried out with primary antibodies against epithelial cadherin (E-cadherin; ab152102), α-smooth muscle actin (α-SMA; ab21027), vimentin (ab188499), MAGEA4 (ab76177), tubulin (as loading control; ab6046) (all from Abcam, Cambridge, UK), or TWIST1 (MBS9382864; MyBioSource San Diego, CA, USA), respectively. The proteins were detected by enhanced chemiluminescence (ECL Plus; Amersham Pharmacia Biotech, Piscataway, NJ, USA)26.

Luciferase Activity Assay

The 3′-untranslated region (3′-UTR) segment of the wt TWIST1 gene, containing the miR-539 binding site, was amplified through PCR and inserted into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA), which was referred to as TWIST1-wt. Site mutation on the miR-539 binding site in the TWIST1-wt, which was referred to as TWIST1-mutant (TWIST1-mut), was obtained using the Directed Mutagenesis System (Invitrogen). TE3 cells were cotransfected with TWIST1-wt or TWIST1-mut and miR-539 or miR-NC using Lipofectamine 2000 reagent. The luciferase activity was analyzed at 48 h posttransfection using the Dual-Luciferase Reporter Assay System (Promega)27.

RNA Extraction and qRT-PCR

Total RNA was extracted with TRIzol reagent (Invitrogen). For the measurement of miR-539 level, RNA (500 ng) was polyadenylated and reverse transcribed to cDNA using an NCode miRNA First-Strand cDNA Synthesis Kit (Invitrogen). cDNA acted as the template for the real-time PCR using FastStart Universal SYBR Green Master (Roche, Indianapolis, IN, USA). For the detection of mRNA expression, Multiscribe RT Kit and SYBR Green PCR Master Mix (both from Applied Biosystems, Foster City, CA, USA) were utilized for the reverse transcription and quantitative PCR, respectively. Real-time PCR was performed with the Applied Biosystems real-time detection system, and the thermocycling parameters were 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 60 s. Melting curve analysis was performed to confirm the specificity of the PCR products. The replicates were then averaged, and fold induction was determined by a ΔΔCT-based fold change calculation28. Each sample was run in triplicate and was normalized to U6 short nuclear RNA (snRNA) levels (for miR-539 level) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; for mRNA levels).

Statistical Analysis

All experiments were repeated three times. The results were presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 5 software (GraphPad, San Diego, CA, USA). The p values were calculated using the one-way analysis of variance (ANOVA) or two-tailed unpaired t-test. A value of p < 0.05 was considered as significantly different.

RESULTS

miR-539 Overexpression Inhibits EMT of TE3 Cells

In this part of the study, miR-539 mimics were transfected into TE3 cells. Then real-time PCR was performed to measure the expression levels of miR-539, and its results showed that miR-539 was markedly upregulated after transfection with the miR-539 mimic compared with the miR-NC group (p < 0.001) (Fig. 1A). Transfected TE3 cells were harvested to measure the mRNA and protein expression levels of E-cadherin, α-SMA, and vimentin by real-time PCR (Fig. 1B) and Western blotting (Fig. 1C and D), respectively. The results showed that the overexpression of miR-539 inhibited the expression of α-SMA and vimentin but elevated the expression of E-cadherin in both mRNA (p < 0.05 or p < 0.01) and protein (p < 0.01 or p < 0.001) expression levels when compared to the miR-NC groups. All the results suggested that overexpression of miR-539 inhibited EMT of TE3 cells.

Figure 1

Effect of microRNA (miR)-539 on the epithelial–mesenchymal transition (EMT) of esophageal cancer cell. (A) miR-539 was overexpressed by transfection of miR-539 mimics. (B) Detection of epithelial cadherin (E-cadherin), α-smooth muscle actin (α-SMA), and vimentin expression in mRNA levels by quantitative reverse transcription (qRT)-PCR. (C, D) Detection of E-cadherin, α-SMA, and vimentin expression in protein levels by Western blot assay. Tubulin was used as a loading control. Data are presented as mean ± standard deviation (SD). *p < 0.05; **p < 0.01; ***p < 0.001. miR-NC, scrambled negative control RNA.

miR-539 Directly Regulates the Expression of TWIST1 in TE3 Cells

The diagram in Figure 2A illustrates the miR-539 base pair with the 3′-UTR of TWIST1. miR-539 mimic or miR-NC and TWIST1-wt or TWIST1-mut were cotransfected into TE3 cells, and then the luciferase activities were measured as shown in Figure 2B. After cotransfection with TWIST1-wt, luciferase activity was significantly reduced by cotransfection with miR-539 mimic compared to that with the miR-NC group (p < 0.001). However, the difference between groups cotransfected with TWIST1-mut was nonsignificant. Then miR-539 mimics or miR-NC was transfected into TE3 cells, followed by Western blot analysis (Fig. 2C and D). The results showed that the protein expression of TWIST1 was observably downregulated by miR-539 overexpression compared with the miR-NC group (p < 0.001). These results indicated that TWIST1 was a target of miR-539.

Figure 2

Effects of miR-539 on TWIST1 expression. (A) miR-539 sequence and 3′-untranslated region (3′-UTR) of Twist-related protein 1 (TWIST1). (B) Luciferase activities after cotransfection of miR-NC or miR-539 mimic and TWIST1-wt (pmirGLO vector carrying the miR-539-binding site of wild-type TWIST1 3′-UTR) or TWIST1-mut (mutant TWIST1-wt). (C, D) TWIST1 expression after miR-539 upregulation by Western blot analysis. Tubulin was used as a loading control. Data are presented as mean ± SD. ***p < 0.001; ns, p > 0.05. hsa, Homo sapiens.

miR-539 Overexpression Inhibits EMT of TE3 Cells by Downregulating TWIST1 Expression

TE3 cells were transfected with different miRNAs or along with plasmids to aberrantly express miR-539 and TWIST1. The cells were harvested and subjected to immunoblot analysis, and the results showed that the miR-539 overexpression-induced alterations of E-cadherin, α-SMA, and vimentin were all reversed by TWIST1 upregulation (p < 0.05 or p < 0.01) (Fig. 3A and B). Thus, we concluded that miR-539 inhibited EMT of TE3 cells by downregulating TWIST1 expression.

Figure 3

Connection of miR-539 and TWIST1 on EMT regulation. (A, B) Protein levels of E-cadherin, α-SMA, and vimentin. Tubulin was used as a loading control. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. pB-NC, empty pBABE-puro vector; pB-TWIST1, pB-NC carrying full-length wild-type TWIST1 gene.

miR-539 Regulates MAGEA4 Expression Through TWIST1 in TE3 Cells

Results of the qRT-PCR in Figure 4A showed that the mRNA level of MAGEA4 was significantly downregulated by TWIST1 knockdown compared with the sh-NC group (p < 0.001). Similarly, the protein expression level of MAGEA4 was also downregulated by TWIST1 knockdown compared with the sh-NC group (p < 0.01) (Fig. 4B and C). Next, cells were stably transfected with pB-TWIST1 or pB-NC and then transiently transfected with miR-NC or miR-539 mimics. The cells were harvested and subjected to immunoblot analysis (Fig. 4D and E). The results showed that miR-539 overexpression remarkably decreased the expression of MAGEA4 compared with the miR-NC group (p < 0.001), while the downregulation was reversed by TWIST1 overexpression compared with the miR-539 mimic + pB-NC group (p < 0.001), indicating that miR-539 could regulate MAGEA4 expression through TWIST1.

Figure 4

Effects of miR-539 and TWIST1 on melanoma-associated antigen (MAGE) A4 expression. (A) MAGEA4 mRNA expression after TWIST1 knockdown. (B, C) MAGEA4 protein expression after TWIST1 knockdown. (D, E) Immunoblot analysis of MAGEA4 after cotransfection. Tubulin was used as a loading control. Data are presented as mean ± SD. **p < 0.01; ***p < 0.001. sh-NC, pSicoR vector carrying a nontargeting short hairpin RNAs; sh-TWIST1, pSicoR vector carrying short hairpin RNAs targeting TWIST1.

MAGEA4 Positively Regulates EMT in TE3 Cells

First, TE3 cells were transfected with sh-MAGEA4, and the results in Figure 5A demonstrated that the protein expression of MAGEA4 was obviously downregulated by sh-MAGEA4 compared with the sh-NC group. Then the transfected cells were harvested and subjected to immunoblot analysis, by which α-SMA and vimentin were found to be downregulated, while E-cadherin was upregulated by MAGEA4 knockdown compared with the sh-NC group (Fig. 5B). Next, cells were stably transfected with pB-MAGEA4 or pB-NC, along with transient transfection with miR-539 mimic or miR-NC. The immunoblot analysis of transfected cells in Figure 5C and D revealed that miR-539 overexpression obviously upregulated E-cadherin (p < 0.01) but downregulated α-SMA (p < 0.05) and vimentin (p < 0.01) when compared to the miR-NC group. However, the alterations induced by miR-539 overexpression could be reversed by MAGEA4 overexpression compared with the miR-539 mimic + pB-NC group (p < 0.01 or p < 0.001). Collectively, all the results suggested that MAGEA4 positively regulates EMT in TE3 cells, and miR-539 overexpression inhibited EMT through downregulating MAGEA4.

Figure 5

Effect of MAGEA4 on EMT of esophageal cancer cells. (A) MAGEA4 protein expression after transfection with sh-NC or sh-MAGEA4. (B) Expression of E-cadherin, α-SMA, and vimentin after MAGEA4 knockdown. (C, D) Detection of E-cadherin, α-SMA, and vimentin expression in protein levels by Western blot assay. Tubulin was used as a loading control. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. sh-MAGEA4, pSicoR vector carrying short hairpin RNAs targeting MAGEA4; pB-MAGEA4, pB-NC carrying full-length wild-type MAGEA4 gene.

DISCUSSION

The incidence of esophageal cancer has increased rapidly in the past decades and caused high rates of mortality for the poor prognosis29,30. ESCC is the main histological type of esophageal cancer with high mortality around the world, which is highly invasive, rapidly metastatic, and results in a poor postoperative quality of life31. In our study, we focused on the effect of miR-539 on ESCC cells and interestingly identified that miR-539 functioned as a tumor suppressor and inhibited EMT by regulating TWIST1–MAGEA4. Subsequent experiments illustrated that TWIST1 was a target of miR-539, and miR-539 functioned through downregulation of TWIST1. Next, miR-539 was demonstrated to regulate MAGEA4 expression, mediated by TWIST1. Finally, MAGEA4 knockdown could repress EMT, and miR-539 functioned through TWIST1-mediated regulation of MAGEA4.

The development and progress of ESCC are influenced by many factors, multiple genes, and progress of many stages32,33. The study of Xue et al. confirmed that ESCC patients with higher miR-483-5p levels had a significantly shorter overall survival time, suggesting that miR-483-5p might be a tumor promoter of ESCC1. However, the functional role of miR-539 in ESCC remains unclear. E-cadherin is a major hemophilic cell–cell adhesion molecule, which inhibits cell migration34. EMT was evaluated as the loss of E-cadherin and the increase in mesenchymal markers, such as α-SMA and vimentin35. In our study, we observed that miR-539 could inhibit EMT in TE3 cells, showing marked upregulation of E-cadherin and downregulation of α-SMA and vimentin, which was consistent with previous literature13,36.

miRs normally participate in physiological activities through the posttranscriptional modulation of target genes. According to the bioinformatics screen with TargetScan, TWIST1 was a potential target gene of miR-539. In our study, the results of the luciferase activity assay strongly validated that TWIST1 was a direct target of miR-539. The following experiments that measured the expression level of TWIST1 in TE3 cells with aberrant expression of miR-539 demonstrated that TWIST1 was negatively correlated with the miR-539 level. In addition, the Western blotting results of cotransfected cells implied that the miR-539 overexpression repressed EMT through downregulating TWIST1, as the TWIST1 overexpression could reverse the changes induced by miR-539 overexpression. To further explore the underlying mechanism of the miR-539-associated modulations, the possible responsive factor of TWIST1 was studied. MAGEA4 has been reported to be upregulated in different types of cancer including ESCC, and the upregulation of MAGEA4 is expected to be a prognostic marker for patients with ESCC37. On account of a previous study, demonstrating that MAGEA4 could be upregulated by TWIST121, we further explored the potential association between TWIST1 and MAGEA4 in TE3 cells. In our study, MAGEA4 was positively correlated with TWIST1 expression, consistent with the results described above. Additionally, MAGEA4 expression was decreased by miR-539 overexpression. We hypothesized that the downregulation of TWIST1 induced by miR-539 overexpression might be the possible reason. The following experiments showed that TWIST1 overexpression could reverse the miR-539-induced alteration of MAGEA4, consolidating our hypothesis. Thus, we concluded that TWIST1 acted as a mediator to connect miR-539 to MAGEA4. Further studies also proved that MAGEA4 knockdown could inhibit EMT, and miR-539 overexpression inhibited EMT through TWIST1-mediated downregulation of MAGEA4.

Taken together, our results collectively showed the roles of miR-539 in ESCC, which are closely correlated with TWIST1–MAGEA4, suggesting that miR-539 can serve as a potential therapeutic target for ESCC.